Immunoassay technique using histidine tags, metals, and chelating agents

ABSTRACT

Two amino acid sequences are joined together using an electron acceptor moiety and a linking moiety, such as a chelating agent. In particular, an amino acid sequence specific for binding to a material interest is linked to an enzyme which acts on an indicator, such as a colorimetric, phosphometric, fluorometric or chemiluminescent substrate. The linking composition is useful in immunoassays.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation in part of application Ser. No.08/360,360, filed Dec. 21, 1994.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to a technique for joining twoamino acid sequences together. The invention has particular utility inlinking an amino acid sequence specific for binding to a material ofinterest to an enzyme which acts on an indicator, such as acolorimetric, phosphometric, fluorometric, or chemiluminescentsubstrate.

2. Background Description

Enzyme linked immunosorbent assays (ELISAs) are widely used methods fordiagnosing an ever increasing number of disease states in man andanimals, especially those diseases which are characterized by thepresence of specific antibodies in the serum. Some common examples ofthese types of diseases include hepatitis B virus (HBV), hepatitis Cvirus (HCV), and human immuno-deficiency viruses (HIV) in humans, andfeline immuno-deficiency virus (FIV) and equine infectious anemia virus(EIAV) in animals.

These methods are based on the simple principle of adsorbing an antigenonto a solid phase, and then using the solid phase antigen to capturespecific antibodies from a host serum. The captured antibodies aresubsequently detected by a variety of methods. The most common method ofdetection uses an enzyme labeled species specific anti-globulin. Asecond, but less commonly used, method is to couple an enzyme directlyto an antigen which is the same antigen as is bound to the solid phaseand then use the enzyme labeled antigen to detect the bound antibody. Inthese systems, the most widely used enzymatic labels are horse radishperoxidase and alkaline phosphatase.

Enzyme linked anti-globulin is often used for detection since the samelabeled reagent can be used in a variety of assays. The use of theenzyme labeled anti-globulin also results in some amplification of thedetection signal since more than one anti-globulin can be bound to eachcaptured antibody or immunoglobulin.

The use of enzyme labeled anti-globulin as the detection reagent alsointroduces some problems. First, the samples to be assayed must bediluted in order to prevent or minimize the non-specific binding of theantibodies or immunoglobulins to the captured antigen. The non-specificbinding produces a high background in the assay and a correspondingdecrease in the sensitivity of the assay. Second, the antigen which isbound to the solid phase must be pure since there is no specificity inthe detection step. Any impurities in the bound antigen would captureunrelated antibodies and would lead to false positive results.

The use of the second method, with an enzyme labeled antigen as thedetection reagent is advantageous in that there is no need for dilutionof the serum samples, since only antibodies which are specific for thebound antigen will bind to the enzyme labeled antigen. Moreover,although this method lacks the inherent signal amplification whichoccurs when enzyme labeled anti-globulin is used, the apparent decreasein sensitivity is offset by the fact that no initial dilution of thesample is necessary.

However, the use of enzyme labeled antigen also has disadvantages, inparticular, a different labeled antigen is required for each assay, orin other words for each detection assay requires a specific antigen.Furthermore, it is generally necessary to develop a specific labelingmethodology for each antigen. Moreover, the direct labeling of theantigen can be difficult, especially if the antigen is either not stableor not reactive under the labeling conditions. It has also been foundthat the coupling methods which are currently used to link enzymes toantigens have produced an alteration in the antigenicity, which is thesubject of the assay.

There is no current method of coupling an enzyme to a protein whichovercomes the disadvantages discussed above. In particular, the currentmethods of antigen labeling can affect the protein characteristicsand/or properties. In addition, a different coupling strategy isrequired for each antigen or protein which is to be labeled.Furthermore, certain proteins cannot be labeled at all or are verydifficult to label. Finally, the label is often not stable and canbecome separated from the protein if the protein-enzyme conjugate issubjected to harsh conditions. Therefore, it is desirable to have acomposition for coupling an enzyme to a protein in which the linkingcomposition can be used for all proteins or amino acid sequences and isstable even under harsh conditions. Furthermore, a method for coupling aprotein or amino acid sequence to an enzyme which does not alter thecharacteristics or harm the protein is desirable.

Several techniques have been developed which utilize recombinantmethodologies to purify proteins from samples using tagged proteins orthe specific interaction between the enzyme-antibody complex and metalions.

One such technique uses "molecular tagging" procedures to purifyproteins in a single step. This tagging method involves the cloning of ashort stretch of polyhistidine onto either the amino or carboxy terminusof the protein. The resultant protein, which has a high affinity fornickel ions, is then purified with a nickel affinity column. Theaffinity of polyhistidine for nickel is great enough that binding occurseven in chaotropic agents such as 6M urea or guanidine hydrochloride.

Another technique is described in U.S. Pat. No. 5,266,686 to Sorenson inwhich a method for isolating an enzyme-antibody conjugate from asolution which contains both the conjugated and unconjugated enzymeusing Ni²⁺ ions bound to a stationary phase. The enzyme-antibodyconjugate binds to the Ni²⁺ ions while the free enzyme does not.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a newtechnique for joining two amino acid sequences together, andparticularly to provide a new technique for joining an amino acidsequence which specifically binds to a material of interest togetherwith an enzyme which can cleave a detecting reagent, such as acolorimetric reagent, chemiluminescent reagent, fluorometric reagent,phosphometric reagent, or the like.

It is another object of this invention to provide novel compositionsuseful in immunoassays where the compositions include an amino acidsequence which binds to a material of interest joined to an enzyme thatcan act on a detecting reagent, wherein the two components of thecomposition are joined together by an electron acceptor moiety, such asa metal ion or the like, that interacts with one amino acid sequence,and a linking moiety, such as a chelating agent or the like, whichinteracts with the enzyme.

It is also an object of this invention to provide a composition whichcan detect antibodies which are present in low amounts.

It is another object of this invention to provide a composition forlinking an enzyme to a protein which can be used on all proteins anddoes not modify the protein.

According to the invention, two amino acid sequences are connectedtogether by an electron acceptor moiety associated with one of the aminoacid sequences and a linking moiety associated with the other amino acidsequence. Particularly good results are obtained when the electronacceptor is a metal ion and the linking moiety is a chelating agent. Ina specific application of this invention related to immunoassays,enzymes and proteins are joined together. Experiments have demonstratedthat the coupling of enzymes to recombinant proteins using moleculartagging methodology can be used in direct sandwich immunoassays todetect antibodies. It has been found that this method of couplingincreases the detection of antibodies in serum. Furthermore, thecomposition can be used to label all proteins. In addition, although thelabeling can occur under mild conditions, the enzyme amino acid orprotein conjugate can also withstand harsh conditions and therefore canbe used in a variety of applications which require an enzyme labeledprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIGS. 1A-1B illustrate a gel obtained from blotting tagged proteins.

FIG. 1A is an illustration of the stained gel obtained from blottingpurified proteins and whole cell extracts which contain tagged protein;and

FIG. 1B shows the binding of α-N,N bis carboxymethyl!lysine (CM-lys)modified peroxidase to the gel.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

It has been discovered that an enzyme can be coupled to an amino acidsequence by an organic chelator and metal ion and that this method ofcoupling can be used with numerous amino acid sequences including avariety of proteins and in particular, antigens. In addition, it hasbeen found that the novel composition for linking the enzyme to theantigen does not affect the properties or characteristics of theantigen.

In a specific application, the composition of the present inventionutilizes an organic chelator and a metal ion to label a protein with anenzyme. An organic chelator, such as nitrilotriacetic acid (NTA),iminodiacetic acid (IDA), bicinchoninic acid (BCA) orN,N,N'-tris(carboxymethyl)ethylenediamine (TED), is coupled to anenzyme. The organic chelator is then charged with a metal ion. Thiscomplex can then be mixed with any protein which contains a histidine"tag" so that the enzyme is coupled to the histidine tagged protein.

As a specific example, an organic chelator, in this case a compound withan NTA functional group, CM-lys, is synthesized using published methodsfrom epsilon amino blocked lysine reacted with bromoacetate, as is shownbelow. ##STR1##

In the practice of the invention, any compound with the appropriatefunctional group can be used including tyrosine or cysteine with an NTAfunctional group and all of the chelating ligands discussed supra.

The CM-Lys is then bound to an enzyme, such as horse radish peroxidaseor to a protein. However, it is possible to use other enzymes such asalkaline phosphatase, beta galactosidase or luciferase. Other proteinssuch as phycobiliproteins could also be used.

If horse radish peroxidase is used the enzyme can be bound through theepsilon amino group using reductive alkylation with periodate activatedperoxidase, making the following bond. ##STR2##

However, it is anticipated that other methods of coupling and otherenzymes and organic chelators can be used. The key feature in the firststep is to join an amino acid sequence, such as an enzyme or the like,together with a linking moiety.

Continuing with the exemplary case where the linking moiety is anorganic chelator such as CM-Lys, the next step is to add a suitableelectron acceptor that can be bound to the linking moiety. Aparticularly preferred electron acceptor is Ni²⁺. However, it isexpected that other metal ions such as Cu²⁺ or Zn²⁺ can also be used.

In the example, to charge the organic chelator, nickel chloride is addedand then dialyzed to form the Ni-NTA-peroxidase, as is shown below.##STR3## This complex of an organic chelator, NTA, a metal ion, Ni andan enzyme, horse radish peroxidase is then mixed with any protein oramino acid sequence which contains a histidine tag. A histidine tag is asequence of three or more adjacent histidines which are generally on thecarboxy or amino terminus end of the protein or amino acid sequence.However, it is expected that any accessible sequence of histidines couldbe used within the practice of the invention. Histidine tags can beadded to the protein or amino acid sequence during the cloning of theprotein or amino acid sequence. The polyhistidines on the protein oramino acid sequence bind very tightly to the Ni-NTA-peroxidase complexproducing a composition which can be used for a variety of systems whichrequire the binding of a protein to an enzyme or to another protein.

An important feature of the above example is that a portion of a secondamino acid sequence is joined to the first amino acid sequence byinteracting with the electron acceptor moiety (metal ion, etc.) which isjoined to the linking moiety (NTA functional group, etc.), which itselfis joined to the first amino acid sequence (enzyme, protein, etc.).

The following examples demonstrate the beneficial effects of using anorganic chelator-metal ion complex as a linker between an enzyme and aprotein and its use in diagnostic assays for antibodies againstimmunoassays of hepatitis B, including HBcAG and HBsAG, hepatitis C, andretro viruses, including HIV, EIAV, and FIV and other related conditionswhich can be diagnosed by the presence of antibodies in humans andanimals.

EXAMPLE 1

This Example details the experimental results which demonstrate theability of the linker complex to specifically bind to the histidinetagged proteins.

Materials and Methods: Nitrilotriacetic acid, horse radish peroxidase,chloronaphthol, hydrogen peroxide, sodium cyanoborohydride, and periodicacid, were obtained from Sigma (St. Louis, Mo.). Microtitre plates,manufactured by Coming, were obtained from Fisher (Columbia, Md.). TMBlue was obtained from TSI (Milford, Mass.).

Preparation of α-N,N bis carboxymethyl!lysine (CM-lys).ε-N-benzyloxycarbonyl lysine (ε-Z-lys) was obtained from AdvanceChemtech (Louisville, Ky.); bromoacetic acid was obtained from Sigma(St. Louis, Mo.). Carboxymethylation of ε-Z-lys was performed in 2Msodium hydroxide by the slow addition of a 2.5 molar excess ofbromoacetic acid which had been dissolved in 2M sodium hydroxide. Thereaction was allowed to proceed on ice for 4 hours, and then at roomtemperature for 24 hours, and finally at 50° C. for 2 hours. Followingthe reaction, the modified Z-lys was purified by recrystallizationfollowing the addition of HCl to pH 2, and the Z group was removed bycatalytic hydrogenation. The extent of reaction was determined by aminoacid analysis by following the disappearance of the starting amino acid.

Coupling of α-N,N carboxymethyl!lysine to horse radish peroxidase. Horseradish peroxidase (100 mg) was dissolved in 25 ml of 50 mM phosphatebuffer, pH 6.8. Sodium periodate was added to a final concentration of0.02M and allowed to react at room temperature for 20 min., and then theenzyme solution was dialyzed overnight against 1 mM acetate buffer, pH4.

CM-lys was then coupled to the enzyme by reductive alkylation asfollows. CM-lys was added to the activated horse radish peroxidase to afinal concentration of 20 mM, in borate buffer, 50 mM pH 8.8, and 10 mMsodium cyanoborohydride. The reaction was allowed to proceed for 4 hoursat room temperature. Nickel sulfate was added to a final concentrationof 50 mM and excess reagents were removed by extensive dialysis againstwater. The modified peroxidase was stable for months at 4 ° C., but wasroutinely stored frozen in small aliquots. The peroxidase activity ofthe enzyme was unaffected by this procedure, as determined by itsspecific activity when TM blue was used as substrate.

Measurement of Nickel Content. The concentration of nickel in the CM-Lysmodified horse radish peroxidase was determined by atomic absorption at232 nm with an Instrumentation Laboratory, Inc., model Video 22 atomicabsorption spectrometer. A series of standards ranging in concentrationfrom 0 to 5.0 μg/ml was used to establish the relationship betweenabsorbance and nickel concentration.

Recombinant Proteins. Table 1 summarizes the recombinant proteins usedin these studies.

                  TABLE 1                                                         ______________________________________                                                       HISTI-                                                                        DINE                                                                 LENGTH   TAG                                                            PRO-  amino    POSI-                                                          TEIN  acids    TION     PHYSICAL PROPERTIES                                   ______________________________________                                        HBV   181      untagged Product of the translation of nucleotides             core                    1903-2450 of the ayw subtype of HBV;                                          soluble, macromolecular particle; 26 nm                                       in diameter; probably 180 subunits                    HBV   157      carboxy  Product of the translation of nucleotides             trun-          terminal 1903-2349 of the ayw subtype of HBV,                  cated                   plus codons for 6 terminal histidine                  core                    residues; soluble, macromolecular                                             particle; indistinguishable from the                                          native core protein by electron                                               microscopy, although the carboxy                                              deleted and the histidine has been                                            added.                                                HCV   180      carboxy  Product of the translation of a                       core           terminal chemically synthesized gene coding for                                        a protein of the same sequence as                                             nucleotides 1-552 of the HCV; poorly                                          soluble in the absence of chaotropic                                          agents, soluble in 6 M urea; monomeric                                        under these conditions.                               HCV   450      amino    Product of the translation of nucleotides             NS#3           terminal 3616-4972 of the HCV, plus codons for                 (heli-                  the amino terminal extension of 6                     case)                   histidines, and a short leader sequence                                       coding amino acids; soluble;                                                  monomeric; enzymatically active.                      CAEV           carboxy  Product of the translation of nucleotides             core           terminal 512-1258 of the CAEV; soluble;                                                monomeric.                                            FIV            carboxy  Product of the translation of nucleotides             core           terminal 1033-1713 of the petaluma strain of                                           FIV; soluble; monomeric.                              HBV   153      untagged Product of the translation of nucleotides             pre-S                   2850-155 of the ayw subtype of HBV;                                           soluble; monomeric.                                   HBV   159      carboxy  Product of the translation of the                     pre-S          terminal nucleotides 2850-155, as above, except                                        for the addition of codons for 6 terminal                                     histidine residues; soluble; monomeric.               ______________________________________                                    

These proteins were chosen because of their potential usefulness indiagnostic assays, and also because they vary greatly in their physicalproperties. The proteins include small soluble proteins, large solublemacromolecular proteins and insoluble proteins.

Labeling of Recombinant Proteins with Peroxidase: In general, equimolaramounts of the recombinant protein and CM-lys modified peroxidase weremixed at pH 7.5, and then diluted to a final peroxidase concentration of1 μg/ml in 10% bovine serum albumin (BSA). The coupling step isessentially instantaneous. In order to obtain the maximum sensitivity inan assay, the optimum ratio of the two proteins is determinedempirically by mixing the proteins at several different ratios andcomparing the results to obtain the combination which gives the highestsignal and lowest background.

Immunoassays: ELISAs were performed in standard 96 well microtitreplates (Coming). The plates were coated with antigen (recombinantprotein) at a concentration of 5-20 μg/ml in bicarbonate buffer, pH 9.5for 18 hours at room temperature, and then washed with phosphatebuffered saline (PBS) containing 0.1% tween 20. Plates were air driedand stored a -20 ° C. until used. The ELISAs were performed by adding50-200 μl of serum (undiluted, or diluted in negative serum of the samespecies), and incubating for 30 minutes at room temperature. The plateswere then washed three times with PBS/tween. Peroxidase-conjugatedantigen was added (50 μl per well) and incubated for 15 minutes at roomtemperature. The plate was then washed with PBS/tween, and 50 μl ofsubstrate was added (TM blue). Color was allowed to develop (generally10 minutes), and then the reaction was stopped by the addition of 100 μlof 1N sulfuric acid. The plates were read in a Dynatech microtitre platereader at 450 nm.

Blotting with CM-Lys modified Peroxidase: SDS polyacrylamide gelelectrophoresis was performed according to the method of O'Farrell, P.H., J. Biol. Chem., 250, 4007-4021, and the proteins electrophoreticallytransferred to nitrocellulose membranes as previously described inBurnette, W. N., Anal. Biochem., 112, 195-203. The membranes wereblocked by incubating with a solution ion of 4% BSA in PBS for 30minutes, and then CM-Lys-modified peroxidase was added to a finalconcentration of 1 μg/ml. The membrane was incubated for 4 hours at roomtemperature with shaking, and then washed extensively with PBS tween.Substrate (chloronapthol/peroxide) was added and the color allowed todevelop for 10 minutes, and then the reaction was stopped by washing.

RESULTS: Preparation of the CM-lys modified Peroxidase: Thecarboxymethylation reaction proceeded to completion to yield a-N, Ncarboxymethyl!lysine as judged by the complete disappearance of lysinein the final product upon amino acid analysis. The compound coelutedwith glycine in the system which was used, and also gave a very lowcolor value. Thus the extent of incorporation of this compound into theperoxidase could not be determined directly by amino acid analysis.However, the number of groups was estimated by determining the number ofnickel (Ni) ions bound per mole of peroxidase by atomic absorptionspectroscopy. Based on the value of 40,000 for the molecular weight ofperoxidase, 10-12 nickel ions were bound per molecule of peroxidase.

Binding of CM-lys modified Peroxidase to Histidine tagged proteins. Inorder to determine if the modified peroxidase would bind to histidinetagged proteins, the compound was first used to bind directly to theprotein which had been blotted onto nitrocellulose. FIGS. 1A and 1B showthe results of the blotting of the purified proteins (CAEV core) and thewhole cell (E. coli) extracts containing the tagged protein. FIG. 1Ashows the stained gel, while FIG. 1B shows the binding of the CM-Lysperoxidase to the gel. It is clear that the peroxidase binds the clonedviral protein and not to the normal cellular proteins. The same resultswere obtained with the other proteins. (data not shown)

Table 2 shows that the CM-Lys-peroxidase binds to the purified histidinetagged proteins which have been coated onto microtitre wells.

                  TABLE 2                                                         ______________________________________                                        A.sub.450                                                                             UNMODIFIED                                                            micrograms                                                                            PEROXIDASE       CM-Lys PEROXIDASE                                    peroxidase                                                                            non-tagged                                                                              histidine tagged                                                                         non-tagged                                                                            histidine tag-                           added   protein   protein    protein ged protein                              ______________________________________                                          0 μg/ml                                                                          0.00      0.01       0.02    0.05                                     2.0 μg/ml                                                                          0.01      0.05       0.00    1.60                                     1.0 μg/ml                                                                          0.01      0.03       0.02    0.86                                     0.5 μg/ml                                                                          0.02      0.03       0.01    0.53                                     ______________________________________                                    

It can also been seen from Table 2 that the proteins which lack thehistidine extension do not bind to the CM-Lys peroxidase, andfurthermore, that both the untagged and tagged protein do not bind tothe unmodified peroxidase.

The experimental data clearly shows that the enzyme labeling of theperoxidase is adequate, and that the peroxidase is readily andspecifically bound to histidine tagged proteins.

EXAMPLE 2

This example details the experimental results which demonstrate theability of the composition of the organic chelator, metal ion, enzymeand protein to detect antibodies which are produced in response tovariety of viruses. Examples of different ELISAs using theCM-Lysperoxidase are provided which illustrate the usefulness of thecomposition. In addition, experimental data has been provided to showthe preferred methods for circumventing some common issues regarding thebinding of unbound peroxidase directly to wells coated with a histidinetagged protein which can produce false positive results.

Assay format 1. It is preferred that the antigen is available as both anon-tagged and a histidine tagged protein. This enables the non-taggedprotein to be used as the capture antigen so that any free peroxidase isprevented from binding to the antigen which is bound to the solid phase.

The following is an illustration of this assay format using thehepatitis B virus (HBV) pre-S protein. The cloning and isolation of thisprotein has been previously described in Delos, S., Villar, M. T., Hu,P., and Peterson, D. L. Biochem. J. (1991) 276, 411-416, which is hereinincorporated by reference. The protein is expressed as a soluble proteinand approximately 50 mg of protein are obtained per liter of cultureusing standard protein isolation methods. This protein was used to coatmicrotitre plates at a concentration of 10 μg/ml. In this experiment,the plates were coated in 20 mM citric acid, pH 3. The pre-S gene wassubsequently modified to include codons for a carboxy terminal extensionof six histidine residues, and then cloned into the same expressionvector (pET 3d). The incorporation of the histidine tag did not affectthe expression level or physical properties of the protein. The soluble,monomeric, histidine tagged protein was labeled with peroxidase bymixing equimolar amounts in Tris/HCl buffer, pH 7.5. The labeled proteinwas diluted in 10% BSA for use in assays.

Table 3 shows the results of assaying rabbit serum for anti-HBsAg pre-Santibodies raised against the non-tagged pre-S protein using the CM-Lysmodified peroxidase.

                  TABLE 3                                                         ______________________________________                                                                  ABSORBANCE AT                                       SERUM       ABSORBANCE AT 450 NM (E. coli                                     DILUTION    450 NM (pure pre-S)                                                                         extract)                                            ______________________________________                                        negative control                                                                          0.02          0.05                                                undiluted positive                                                                        >2.0          >2.0                                                1:10        >2.0          >2.0                                                1:20        >2.0          >2.0                                                1:40        1.3           1.42                                                1:80        0.65          ND                                                  1:160       0.30          ND                                                  1:320       0.13          ND                                                  ______________________________________                                    

The negative serum gave absorbance values of less than 0.05, while thepositive serum gave values of greater than 2. The positive serumremained clearly positive even at a dilution of 1:160 in normal rabbitserum. It can also be seen from Table 3 that the protein does not needto be purified in order to label it, since the labeling reagent,CM-lys-peroxidase, is specific for the histidine tagged protein. It isshown that the use of an E. coli extract containing the histidine-taggedprotein mixed with the CM-lys modified peroxidase produced the sameassay results as with the purified proteins. It is also shown that theprotein which is used on the solid phase to capture the antibodies doesnot need to be pure, since only the specific antibodies are detected bythe peroxidase labeled antigen. However, it should be noted that if thebound protein is contaminated, the sensitivity of the might be lowered,since the contaminant proteins would compete for binding sites of thesolid phase and therefore, lower binding of the desired protein would beexpected.

Assay format 2. It is also possible to use the protein with thehistidine tag for both the capture and the coating protein, particularlyin situations where the protein lacking the histidine tag is notavailable. However, it has been found that if the untagged protein isnot available, a modification in the method, described above, may berequired.

An illustration of assay format 2 is provided below using an assay forantibodies against the HCV NS3 protein, CAEV and FIV core proteins. Inthese experiments, microtitre plates were coated with the purifiedhistidine tagged proteins, and the same proteins were also coupled toperoxidase by mixing equimolar amounts followed by dilution in 10% BSAin Tris/HCl, pH 7.5. To prevent any un-bound peroxidase, or peroxidasewith unsatisfied ligands, from binding directly to the plate, thehistidine tagged protein on the plates was blocked by incubation with300 mM nitrilotriacetate/Ni complex (prepared by mixing equimolaramounts of nitrilotriacetate and nickel sulfate), in Tris/HCI, pH 8.0for 2 hours, followed by washing with PBS tween. In addition, this samecomplex (10 mM, final concentration) was added to the antigen-peroxidaseconjugate solution.

Table 4 shows the results of ELISAs using CM-Lysperoxidase with solidphase histidine tagged proteins and using positive and negative sera.The values reported are the average of the absorbance values at 450 nmof duplicate determinations on two or more different undiluted serumsamples for each.

                  TABLE 4                                                         ______________________________________                                        SOLID PHASE PROTEIN USED                                                      HCV         CAEV     HCV                                                      NS3         core     core     FIV core                                                                             BIV core                                 ______________________________________                                        negative                                                                             0.012    0.04     0.02   0.01   0.02                                   sera                                                                                 0.03     0.02     0.04   0.02                                          positive                                                                             1.65     1.86     >2.0   0.77   1.12                                   sera                                                                                 >2.0     1.7      1.4    1.14   0.83                                   ______________________________________                                    

As noted above, the results provided in Table 4 show that negative seragave very low background, while positive sera gave high absorbance. Thesame method was found to give good results with the HIV core protein.(data not shown) It is also shown that the blocking step with thenickel-NTA complex was effective, since after the blocking step theCM-lys-peroxidase does not bind directly to the ELISA wells.

It is also known that many recombinant proteins are poorly soluble inthe absence of detergents or chaotropic agents such as urea andguanidine. Therefore, as discussed above, it is necessary to provide acomposition to link the enzyme to the protein which can withstand harshconditions. An example of a poorly soluble protein and the ability of ametal ion-organic chelator link to withstand harsh conditions isprovided below.

The hepatitis C virus core protein is an example a protein which is notsoluble at high concentrations in Tris/HCl, pH 7.5. However, it isreadily soluble in 5 M guanidine HCl, pH 7.5. The protein, which as beensolubilized in guanidine, remains in solution when diluted to the lowconcentration used for coating microtitre plates (10 μg/ml), and so isreadily used as the capture antigen in ELISA. Moreover, the antigen isreadily coupled to the derivatized peroxidase by mixing the HCV coreprotein, dissolved in 5M guanidine, with equimolar amounts of peroxidasedissolved in PBS, followed by immediate dilution to the usual peroxidaseconcentration (1 μg/ml). Since the binding of the peroxidase to thehistidine-tagged protein proceeds quickly and the mixture is dilutedimmediately, the peroxidase does not lose its activity.

The results of the assaying of human sera for anti HCV core antibodiesusing this method are shown above in Table 4. The NTA/Ni blocking step,described above, was used in these experiments, since histidine taggedprotein was also used as the capture antigen.

A further issue which must be considered is the accessibility of thehistidine tag, particularly due to the structure of the protein, suchthat the protein will not bind to the CM-lys derivatized peroxidase. Oneexample of this is the hepatitis B core protein (HBcAg), having acarboxy terminal histidine tag. In this case, the histidines are hiddenwithin the particle interior. However, 3M thiocyanate was found toexpose the histidine tag, while not destroying the HBcAg epitopes.

Table 5 shows the results of using a CM-lys-peroxidase conjugated toHBcAg to assay human sera for antibodies against HBcAg. Table 5

    ______________________________________                                        SAM-          RESULTS OF              RESULTS OF                              PLE           PREVIOUS   SAMPLE       PREVIOUS                                NO.   A.sub.450                                                                             ASSAY      NO.    A.sub.450                                                                           ASSAY                                   ______________________________________                                        1     0.00    1.718 (-)  13     0.41  0.152 (+)                               2     0.09    1.650 (-)  14     0.79  0.107 (+)                               3     0.13    1.784 (-)  15     0.69  0.152 (+)                               4     0.10    1.712 (-)  16     0.52  0.149 (+)                               5     1.31    1.442 (-)  17     0.80  0.142 (+)                               6     >2.0    0.267 (+)  18     0.63  0.107 (+)                               7     1.70    0.365 (+)  19     >2.0  0.155 (+)                               8     0.62    0.196 (+)  20     1.56  0.132 (+)                               9     0.88    0.221 (+)  21     0.54  0.142 (+)                               10    0.78    0.185 (+)  22     0.76  0.155 (+)                               11    0.45    0.214 (+)  23     0.96  0.161 (+)                               12    0.63    0.167 (+)  24     0.80  0.137 (+)                               ______________________________________                                    

In this experiment, the capture antigen was the untagged full lengthcore protein. The peroxidase conjugate was prepared by adding equimolaramounts of histidine tagged HBcAg, dissolved in 3M potassium thiocyanateand CM-lys-peroxidase, dissolved in Tris/HCl, pH 7.5, followedimmediately by dilution in 10% BSA in PBS.

Table 5 presents a series of sera which constitute a seroconversionpanel obtained from Serologics, Inc. This panel includes 25 samples ofsera taken from a single infected individual at approximately weeklyintervals during the course of a natural infection with HBV. The valuespresented are the absorbencies observed. It is clear from this data thatthe negative values are very low and that the positive values can beeasily distinguished. The table also provides the results of commercialcompetitive ELISAs for antibodies against HBcAg. The positive andnegative assignments are the interpretations of the values according tothe manufacturer of the assay. It should be noted, as can be seen fromthe data presented in Table 5, assays using the metal ion-organicchelate complex, as described herein, were able to detect theseroconversion one week earlier, at sample 5 rather than sample 6, thanthe commercial assay.

The experimental data, set forth above, clearly demonstrates that CM-lysmodified peroxidase is an effective general labeling reagent for usewith histidine tagged proteins. The composition of an organicchelator-metal ion complex to bind a protein to an enzyme can be used inELISAs to detect antibodies in serum.

It has been shown that the method and composition can be used with bothsoluble and insoluble proteins as well as with pure or impure proteins.Since these proteins are exemplary of broad and diverse classes ofproteins, it is expected that this composition and method will be usefulfor binding most amino acid sequences to a labeling compound.

Furthermore, the availability of numerous bifunctional crosslinkingreagents enables that the organic chelator-metal ion complex can becoupled to any protein or macromolecule, and subsequently used as areporter group to attach to histidine tagged proteins.

The method can also be used in other applications. For example, enzymelabeled proteins can be used to study the interaction between a proteinand cell receptors. Since the labeling method of labeling discussedsupra, enables the enzyme labeling to be specifically targeted to eitherthe carboxy or amino terminus of a protein, it is able to provide aconstruct which is less likely to interfere with the putative bindingsite of the protein.

While the invention has been described in terms of a single preferredembodiment, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

Having thus described my invention, what I claim as new and desire tosecure by Letters Patent is as follows:
 1. An assay method, comprisingthe steps of:combining a labeled amino acid sequence specific for anamino acid sequence of interest in a sample, said labeled amino acidsequence comprisingi) a histidine tag, ii) an enzyme covalently bondedto a chelating agent, and iii) a metal electron acceptor, wherein saidenzyme is linked to said histidine tag by said metal electron acceptorwhich bonds with both said chelating agent and said histidine tag;allowing said labeled amino acid sequence to bind to said amino acidsequence of interest in said sample; and measuring the amount of labeledamino acid sequence bound to said amino acid sequence of interest. 2.The method of claim 1 wherein said step of measuring includes the stepsof:adding an indicator substrate clearable by said enzyme covalentlybound to said chelating agent of said labeled amino acid sequence; andmeasuring a cleaved reaction product derived from said indicatorsubstrate from said enzyme cleaving said indicator substrate.
 3. Themethod of claim 1 further comprising the step of binding said labeledamino acid sequence to a solid phase.
 4. The method of claim 1 furthercomprising the step of binding said amino acid sequence of interest to asolid phase.
 5. The method of claim 1 wherein said step of combining isperformed in the presence of a non-labeled amino acid sequence specificfor said amino acid sequence of interest, said non-labeled amino acidsequence being identical to said labeled amino acid sequence in allrespects except that it lacks said histidine tag, said enzyme covalentlybonded to a chelating agent, and said metal electron acceptor.
 6. Amethod as in claim 1 wherein said amino acid sequence of interest iselicited by Bovine Immunodeficiency Virus.
 7. A method as in claim 1wherein said amino acid sequence of interest is elicited by FelineImmunodeficiency Virus.
 8. A method as in claim 1 wherein said aminoacid sequence of interest is elicited by Caprine Arthritis andEncephalitis Virus.
 9. A method as in claim 1 wherein said amino acidsequence of interest is elicited by Hepatitis C Virus.